Muc2, meprinβ, tjp1). Moreover, EPX altered the composition and abundance of gut microbiota in the colon of mice. The alpha diversity indices  in the gut microbiota increased after exposure to EPX for 28 days. Interestingly, EPX increased the ratio of Firmicutes to Bacteroides and the abundance of other harmful bacteria including Helicobacter and Alistipes. Based on the untargeted metabolomic analysis, it was found that EPX altered the metabolic profiles of the liver in mice. KEGG analysis of differential metabolites revealed that EPX disrupted the pathway related to glycolipid metabolism, and the mRNA levels of related genes were also confirmed. In addition, the correlation analysis showed that the most altered harmful bacteria were associated with some significantly altered metabolites. The findings highlight that EPX exposure changed the micro-environment and lipid metabolism disturbance. These results also suggest that the potential toxicity of triazole fungicides to mammals cannot be ignored.Epoxiconazole (EPX), a triazole fungicide, is widely used in agriculture to control pests and diseases. High residual and occupational exposure to EPX increases health risks, and evidence of potential harm to mammals remains to be added. In the present study, 6-week-old male mice were exposed to 10 and 50 mg/kg bw EPX for 28 days. The results showed that EPX significantly increased the liver weights. EPX also decreased the mucus secretion of the colon and altered intestinal barrier function in mice including a reduced expression of some genes ( For the past few years, large amounts of triazole fungicides have been approved for use in agriculture. With the increasing reports of fungicides, widespread safety concerns have been raised about the whereabouts of residues and the toxic effects of pesticide exposure ,2. FungiDrugs entering the body by intragastric administration are common in vivo experiments of physiology, pathology, and toxicology . The gasWith the development of mass spectrometry, the application of the metabolome in toxicological research has rapidly increased . At enviEPX  and corn oil  were purchased from Aladdin Chemical Reagent Industry  and stored at 4 °C.n = 24) were supplied by the China National Laboratory Animals Resource Center . The mice were randomly assigned to cages . Water and food were freely available throughout the feeding process. After a week of acclimatization, all mice were randomly placed into three treatment groups: Con, EPX-L (10 mg/kg bw), and the EPX-H group (50 mg/kg bw) (n = 8). The 100 μL corn oil with doses of 0, 10, 50 mg/kg bw were administered intragastrically daily, respectively, and the doses were selected based on the LD50 (>5000 mg/kg) of the rat. The duration of the gavage was 28 days, as a sub-chronic exposure test. Each mouse could drink pure water and consume commercial feed freely.The five-week-old male C57BL/6 mice  at 4 °C for 10 min. The killed mice were dissected promptly, and the organs were weighed and immediately placed in liquid nitrogen. The serum and rapid-freezing organs were deposited at −80 °C for further testing. All mouse experiments were authorized by the Guiding Principles of Zhejiang University of Technology (20211117091).n = 4) selected from the Con and EPX-H treatment group were randomly used for histological analysis. Briefly, the liver and colon were fixed in 4% paraformaldehyde overnight, embedded within paraffin wax, and cut into 5 µm-thick sections. The 5 µm-thick sections of livers were stained with hematoxylin and eosin (H&E), and the sections of the colon were stained with Alcian blue-periodic acid Schiff (AB-PAS). ImageJ2 was used for the quantitative analysis of AB-PAS staining.The liver and colon  primary antibody was applied at 1:500, then stained with FITC-conjugated  anti-rabbit secondary antibody. For the immunofluorescence analysis, the rabbit anti-claudin-1  and -ZO-1  primary antibodies were applied at 1:500, then stained with the HRP-conjugated  anti-rabbit IgG secondary antibody. All images were captured with a fluorescence microscope .g for 10 min. The supernatant was used to assess the biochemical indicators. The above tests followed the instructions in the commercial kits purchased from Nanjing Jiancheng Institute of Biotechnology . In addition, the protein concentration in the liver was detected with a Bradford Protein Assay Kit  for the relative quantification of each indicator.The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated to assess liver function. The serum and hepatic triglyceride (TG), total cholesterol (TC), glucose (Glu), pyruvate (PYR), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured to evaluate the changed biochemical indices of the mice. The serum samples were directly used to detect the liver function and biochemical indicators. Liver samples were processed by adding PBS at 1:9 for grinding and centrifuging at ‘1000× The total RNA was extracted from the liver and colon of mice with the TRIZOL reagent. All extraction steps followed the instructions, and chloroform, isopropyl alcohol, and 75% alcohol were prepared. A Nano-300  detected the RNA concentration and purity. Furthermore, 1000 ng RNA was used for the cDNA synthesis using a reverse transcription kit. The next step was RT-qPCR, which was performed with the SYBR green system. All reagents were purchased from Vazyme , and all operations were conducted according to the instructions. Here, the specific primer sequences of the gene are listed in ® DNA PCR-Free Sample Preparation Kit and adding index codes. The Qubit@ 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system evaluated the library quality. The Illumina NovaSeq platform was used for high-throughput sequencing, and the 250 bp paired-end reads were obtained.The V3–V4 region of 16S rRNA sequencing was used to explore the intestinal microflora changes between the Con and EPX-H treatment groups. The total genomic DNA was extracted from the colon contents using a magnetic bead extraction method in our previous study . Then, tAfter data splitting and screening, sequence analysis was conducted with Uparse software (Uparse v7.0.1001) . For theThe microflora differences at the phylum level were determined by qPCR with DNA, and the primers are shown in 2O]. After sanding , the homogenate was treated with ultrasound for 30 min and placed on ice for 30 min before being centrifuged at 12,000 rpm for 10 min at 4 °C to achieve the supernatant for concentration and drying. The samples were redissolved using 50% acetonitrile solution with 2-chloro-L-phenylalanine solution , which was used for LC-MS detection. The detection included chromatography conducted by the ultra-performance liquid system  and mass spectrometry was performed by the mass spectrometer detector . The above extraction, detection, and analysis work were all supported by Suzhou PANOMIX Biomedical Tech Co., Ltd. . Details are presented in the The livers were used for metabolomics analysis based on LC-MS. A sample of 100 g of liver tissue was ground in tissue extract [75% : 25% Hp ≤ 0.05) and ** (p ≤ 0.01).Data were executed with GraphPad Prism 7.0 software and presented as the mean ± standard error of the mean (SEM). Data analysis between the Con and treatment groups was performed with one-way ANOVA followed by Dunnett’s post hoc test. Statistical significance was denoted with * (p < 0.05), with the increase in concentration as well as the ratio of liver to body weight (p < 0.05) in a concentration-dependent manner. Similarly, the fat weight did not vary, and the ratio of fat to body weight decreased (p = 0.035) in the EPX-H group.After continuous EPX administration for 28 days, the body weights of mice showed an upward trend. However, there was no statistical significance A. Fastedy weight B. AlthouIn addition, there was no apparent pathological damage (vacuolization) to the liver of mice after being exposed to EPX C. The sep < 0.05) with the concentration dependence, and the TG and Glu levels did not change. The levels of PYR decreased (p = 0.03) in the EPX-L treatment group. Furthermore, HDL (p = 0.005) and LDL (p = 0.03) were lower in the high-concentration treatment group than in the other groups.For the serum biochemical indices , the TC p = 0.03) in the EPX-H treatment group. The TG, Glu, and PYR levels did not change after exposure to EPX. The HDL and LDL were not detected in the liver (not shown). Furthermore, we found that the NEFA level also decreased (p = 0.02) in the liver of mice exposed to EPX-H.In addition, the hepatic biochemical indices were also detected . The TC Muc2 in the colon of mice treated with EXP-L was significantly reduced (p = 0.026). Additionally, the transcriptional level of meprinβ was decreased in all EPX treatment groups (p = 0.016).The mucus secretion of mice showed a numerical decrease in the EPX-H group compared to the Con group . Immunohp = 0.040) and EPX-H (p = 0.060) group when compared to the Con group, and the mRNA level of ZO-1 was also decreased in the EPX-H group (p = 0.059).In addition, immunofluorescence revealed no significant changes in the claudin-1 and ZO-1 protein expression C,D. Similyz, plazr4a, and ang4 were all increased significantly (p < 0.05) in the EPX-H treatment group. The other genes  did not change significantly.As shown in cftr, nkcc1, slc26a3, slc26a6), only the mRNA level of slc26a3 increased in a concentration-dependent manner (p < 0.05).Additionally, we analyzed the relative mRNA levels of genes involved in ionic transport in the colon of mice . The resp = 0.069) of the two treatment groups. The Shannon and Simpson indices increased significantly (p < 0.05) in the EPX treatment group. Two other indices  also increased, though not significantly. The above results show that EPX exposure increased the intestinal microbiota diversity in the colon content of mice. In addition, EPX disrupted the intestinal microbiota composition at the phylum level  in the EPX-H treatment group. Although our RT-qPCR showed that only the relative abundance of Firmicutes declined (p = 0.03) in the EPX-L treatment group, 16S rRNA sequencing showed that the relative abundance ratio of Firmicutes to Bacteroides (p = 0.02) was significantly increased  and downregulated .Moreover, the difference in the intestinal microbiota in the colon contents between the EPX treatment group and the Con group was evaluated at the phylum level through RT-qPCR A. The rencreased B. LEfSe ficantly C. The re altered D includiAlloprevotella, Prevotellaceae, and Bacteroides , the main component of tight junctions, on the actin cytoskeleton to maintain the integrity of interepithelial connections [tjp1 and zo-1 genes  is widely used for the metabolic profiling of urine, serum samples, or animal tissue extracts . The prelistipes F. We belPPAR-γ, scd1, PPAR-α, MCAD CPT1, acot-1, and fabp1, decreased significantly, which classified into fatty acid synthesis, fatty acid β-oxidation, and fatty acid transport. Reduced expression levels of CPT1, AOX, and MCAD in adult zebrafish exposed to EPX were also confirmed [GK, PK) and Glu transport (Glut2) increased significantly. Our previous experiments on larval zebrafish have also shown that EPX reduced glucose levels and increased the expression of GK and HK1 but decreased the PK expression levels [Some metabolites in glycolipid metabolism changed significantly G. Again,onfirmed . In addin levels . Transcrn levels . It is aThe toxicity of EPX to the liver and gut of male C57BL/6 mice exposed for 28 days was studied with 16S sequencing and untargeted metabolomics. EPX increased the liver weight and decreased the relative kidney weight. EPX also altered the intestinal barrier function and induced gut microbiota dysbiosis. Furthermore, EPX disrupted the metabolic profile of the liver in mice and induced glycolipid metabolism disorder. These results clarify the effect of EPX on the gut, intestinal microenvironment, and liver metabolism of the mice and provide a more theoretical basis for the risk assessment of EPX.


De novo combination of natural product (NP) fragments by means of efficient, complexity- and stereogenic character-generating transformations to yield pseudo-natural products (PNPs) may explore novel biologically relevant chemical space. Pyrrolidine- and tetrahydroquinoline fragments rarely occur in combination in nature, such that PNPs that embody both fragments might represent novel NP-inspired chemical matter endowed with bioactivity. We describe the synthesis of pyrroloquinolines by means of a highly enantioselective intramolecular exo-1,3-dipolar cycloaddition catalysed by the AgOAc/(S)-DMBiphep complex. The cycloadditions proceeded in excellent yields (up to 98%) and with very high enantioselectivity (up to 99% ee). Investigation of the resulting PNP collection in cell-based assays monitoring different biological programmes led to the discovery of a structurally novel and potent inhibitor of the Hedgehog signalling pathway that targets the Smoothened protein. c]quinoline pseudo-natural products by means of a highly enantioselective intramolecular 1,3-dipolar cycloaddition yielded a novel Hedgehog signalling inhibitor chemotype that targets the Smoothened protein.Synthesis of pyrrolo[3,2- In PNP design and synthesis, NP fragments are combined in novel arrangements not found in nature by means of efficient, complexity- and stereogenic character-generating transformations, and for such syntheses enantioselective 1,3-dipolar cycloadditions have proven to be of high value.2Compound classes endowed with pronounced bioactivity are often inspired by natural product (NP) structure and have high stereogenic content, which positively correlates with performance in drug discovery.6 such that PNPs that embody both fragments might represent novel NP-inspired chemical matter that is endowed with bioactivity. We have recently developed asymmetric syntheses of pyrroloquinolines 1  or a Cu(ii) salt led to greatly reduced enantioselectivity , enantioselectivity was lower regardless of the substitution site (N-benzyl substituents or an N-methyl group (R2) led to a decrease of ee to 79–88% , which corresponds to the C6 position in the final quinoline scaffold, increased the enantioselectivity. Diverse substituents with different electronic properties or steric demand were well tolerated and afforded the desired cycloadducts 3 in good yields and with excellent enantioselectivity  spectra13 for 3l, respectively, and were assigned by analogy to the other cycloadducts  on enantioselectivity, diverse substituent combinations were investigated. In the presence of a methyl group at R3, enantioselectivity improved from 75%–85% to 94%–97% for regioisomeric chlorophenyl substituents at R1  may be rationalised by the transition state proposed in S)-DMBiphep and the iminoester intermediate, which would be deprotonated to the azomethine ylide by Cs2CO3. In this transition state, the dipolarophile would preferably approach the 1,3-dipole from the back side to avoid steric repulsion between the substrate backbone and the ligand. In particular, in the presence of a substituent R3, the repulsion would be pronounced, thus leading to higher enantioselectivity. In comparison, no improved enantioselectivity was recorded if a substituent R4 was introduced because this position does not point towards the ligand. We also note that the intramolecular cycloaddition proceeds with an exo approach due to the steric demands and conformational rigidity in the transition state.The direction of the stereoselection and the pronounced influence of the substituent 14 Excessive activity of the pathway is associated with malignancy, such as medulloblastoma,15 basal cell carcinoma16 and rhabdomyosarcoma.17 Therefore, inhibitors of the Hh signalling pathway have emerged as attractive therapeutic options in oncology.18In order to investigate whether the cycloadducts 3 are endowed with bioactivity, they were subjected to cell-based assays monitoring different biological programmes, such as oncogenic signalling, autophagy and immunometabolism. Gratifyingly, the compound collection defines a new inhibitor chemotype of signal transduction through the Hedgehog pathway. The Hedgehog (Hh) signalling pathway plays a critical role in the regulation of embryonic development, post-embryonic tissue homeostasis and regeneration in vertebrates.19 Gratifyingly, most of the compounds showed potent inhibition of Hh signalling  of 0.29 ± 0.05 μM  displayed an approximately 30-fold lower IC50 value of 0.15 ± 0.03 μM. This tendency was also observed in the orthogonal Gli-dependent reporter gene assay in Shh-LIGHT2 cells20 with an IC50 of 0.17 ± 0.02 μM for ent-3a while 3a was inactive . Upon binding, cellular internalisation of Ptch1 relieves the Ptch1-mediated inhibition of the Smoothened (SMO) protein, allowing for SMO translocation to the membrane, which triggers a signalling cascade that ultimately activates transcription of Hh target genes such as Ptch1 and glioma-associated oncogene homolog 1 (Gli1).14 The protein SMO is a clinically validated therapeutic target and many relevant Hh pathway inhibitors act by SMO antagonism, including vismodegib and cyclopamine, which specifically bind to the heptahelical bundle of SMO leading to pathway inhibition.22 To detect whether 3a also binds to SMO, a competitive SMO-binding assay was performed with a BODIPY-labelled derivative of the SMO binder cyclopamine.19 HEK293T cells transfected with a SMO-expressing plasmid were incubated with the compounds and BODIPY-cyclopamine at 5 nM. Less potent compound 3a did not decrease BODIPY-related fluorescence  were processed for the modelling. Only structure 5L7I could differentiate well between 3a and ent-3a according to the docking scores, which suggests that ent-3a may adopt a similar binding mode, as observed for the ligand vismodegib in 5L7I.23 The binding pose suggests that ent-3a occupies a space closer to the entrance of the pocket, while vismodegib resides deeper inside. For this model, the key interactions are hydrogen bonding between the ester carbonyl oxygen of ent-3a to Arg400 and of an amino proton to Gln477 , observed in a relapsed patient with metastatic medulloblastoma after vismodegib treatment.24 It is believed that D473H mutation impairs the tight binding of vismodegib through an indirect pathway.26 The cation–π interaction between Arg400 and the pyridine ring of vismodegib is a key source of high affinity, where Asp473 forms a tight hydrogen bond with Arg400  due to its relatively high degree of saturation 33 were calculated and visualised in a two-dimensional plot to evaluate fragment compositions and drug-like properties, respectively. Ent-3a occupies an unpopulated area that has a high NP-likeness score and high QED score relative to the previously developed SMO antagonists -DMBiphep catalyst, resulting in excellent yields and enantioselectivity. Exploration of the substrate scope revealed that enantioselectivity in the cycloaddition is decisively influenced by the site-specific introduction of a substituent. Biological evaluation of the resulting pseudo-NP collection revealed a novel Hh signalling inhibitor, whose activity was highly dependent on absolute configuration. Compound ent-3a binds to the SMO protein and, thereby, potently suppresses Hh target gene expression to an extent similar to the clinically approved SMO antagonist vismodegib. Cheminformatic analysis of ent-3a and reported SMO antagonists revealed that the pseudo-NP has unique properties and constitutes a new chemotype for SMO inhibition.In conclusion we have developed a new enantioselective intramolecular 1,3-dipolar cycloaddition for the combination of the pyrrolidine- and tetrahydroquinoline-NP fragments. The highly challenging asymmetric cycloaddition was enabled by the use of a AgOAc/(rac-3j have been deposited in the Cambridge Crystallographic Data Centre under accession number CCDC 2243515.All experimental and characterisation data, as well as NMR spectra are available in the ESI.J. L. and R. R. Z. contributed equally. J. L. and H. W. conceived and directed the project. J. L. and S. M. performed the organic synthesis. J. L., R. R. Z., S. P., C. W. and J. F. performed the biological evaluation of the compound. A. K. and C. S. conducted the X-ray crystallography. C. G. and C. M. performed the computation of VCD spectra. J. L. performed molecular docking and the prediction of drug-like properties. A. P. and M. G. carried out the cheminformatic analysis. J. L., R. R. Z. and H. W. wrote the paper. All authors discussed the results and commented on the manuscript.The authors declare no conflict of interest.SC-014-D3SC01240A-s001


Lung point-of-care ultrasonography (L-POCUS) is highly effective in detecting pulmonary peripheral patterns and may allow early identification of patients who are likely to develop an acute respiratory distress syndrome (ARDS). We hypothesized that L-POCUS performed within the first 48 hours of non-critical patients with suspected COVID-19 would identify those with a high-risk of worsening.POCUSCO was a prospective, multicenter study. Non-critical adult patients who presented to the emergency department (ED) for suspected or confirmed COVID-19 were included and had L-POCUS performed within 48 hours following ED presentation. The lung damage severity was assessed using a previously developed score reflecting both the extension and the intensity of lung damage. The primary outcome was the rate of patients requiring intubation or who died within 14 days following inclusion.Among 296 patients, 8 (2.7%) met the primary outcome. The area under the curve (AUC) of L-POCUS was 0.80 [95%CI:0.60–0.94]. The score values which achieved a sensibility >95% in defining low-risk patients and a specificity >95% in defining high-risk patients were <1 and ≥16, respectively. The rate of patients with an unfavorable outcome was 0/95 (0%[95%CI:0–3.9]) for low-risk patients (score = 0), 4/184 (2.17%[95%CI:0.8–5.5]) for intermediate-risk patients (score 1–15) and 4/17 (23.5%[95%CI:11.4–42.4]) for high-risk patients (score ≥16). In confirmed COVID-19 patients (n = 58), the AUC of L-POCUS was 0.97 [95%CI:0.92–1.00].L-POCUS performed within the first 48 hours following ED presentation allows risk-stratification of patients with non-severe COVID-19. The COVID-19 pandemic has developed worldwide since its emergence in December 2019. Many patients have an uncomplicated course with minor symptoms, however, around 4% develop respiratory symptoms and require hospitalization . There iPulmonary computed tomography (CT-scan) appears to be very sensitive (97%) and quite specific for diagnosis of COVID-19 in patients with a clinical suspicion, provided that it is not performed within the first 4 days after symptom onset . Those pLung point-of-care ultrasonography (L-POCUS) is a simple, non-invasive, non-irradiating, inexpensive imaging tool available at the bedside and increasingly used by emergency physicians in their everyday clinical practice. A pre-COVID-19 study showed that L-POCUS is better than chest X-ray for detection of pneumonia and may be an alternative to the CT-scan as a screening and prognostic tool . Indeed,A recent publication has shown good prognostic value of lung damage estimated by L-POCUS at admission in confirmed COVID-19 population .The aim of this study was to determine the performance of L-POCUS at the time of ED presentation or within the first 48 hours in identifying, among patients with confirmed or highly suspected COVID-19, those who are at high-risk of adverse outcomes such as respiratory failure or death.The point-of-care ultrasonography for risk stratification of COVID-19 patients’ study (POCUSCO) was a non-interventional, prospective, multicenter study that was conducted in the ED of 11 hospitals in France and Belgium. This study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by French and Belgian ethics committees, and all participants provided written informed consent. This study adheres to STROBE guidelines .Patients were enrolled if they met all of the following criteria: age ≥ 18 years; typical COVID-19 symptoms and at least one of the three following features: i) positive SARS-CoV-2 RT-PCR, ii) typical CT-scan lesions, iii) highly-suspected COVID-19 based on the in-charge physician judgement; no requirement for respiratory support and/or other intensive care, and not subject to a limitation of care; membership of a social security scheme.2, history of pneumonectomy) were excluded.Patients for whom the follow-up at Day 14 was impossible or who had a condition making L-POCUS impossible   15]..15].The main objective was to assess the ability of L-POCUS to identify COVID-19 patients with a high-risk of unfavorable outcome. The primary endpoint was the development of severe COVID-19 within the 14 days after ED admission defined as a stage ≥ 6 on the WHO-OSCI. This stage relates to a severe inpatient requiring invasive ventilation (stage 6), and/or additional organ support (stage 7), or who died whatever the cause (stage 8). The ability of L-POCUS to predict the primary outcome occurrence was evaluated by the area under the curve (AUC) of the receiver operating characteristic (ROC) curve and its 95% confidence interval (95%CI). A sensitivity analysis was performed with the 14-day all-cause mortality rate as the outcome.The secondary objectives were: 1) To determine the threshold values of L-POCUS to stratify patients into three groups according to their risk of adverse outcome: low-risk, intermediate-risk, and high-risk patients. 2) To assess the impact of adding the result of L-POCUS evaluation to two risk-stratification clinical scores: the quick Sequential Organ Failure Assessment (qSOFA) and the CRB-65 , 17. 3) We finally performed a subgroup analysis in patients for whom the diagnosis of COVID-19 was initially or subsequently confirmed by a positive RT-PCR for SARS-CoV-2.Initial L-POCUS was performed with ultrasound scanners using low frequency (2–5 MHz) transductors. The Bedside Lung Ultrasound in an Emergency (BLUE)-Protocol was applied to patients in erect or semi-recumbent positions depending on dyspnea severity  14]. Ea. Ea14]. Continuous variables were expressed as mean and standard deviation values. Categorical variables were described using numbers, percentages, and their 95%CI. The AUCs and their 95%CI were determined by the .632 bootstrap method. For the primary outcome, we determined in advance that the L-POCUS prognostic value would be considered as clinically relevant with a good level of evidence if the lower bound of the 95%CI of the AUC was equal to or greater than 0.7. To perform risk stratification in three groups of patients with a low, intermediate, or high-risk of an unfavorable outcome, two thresholds were calculated. The first maximized specificity with a sensitivity greater than or equal to 95% and the second maximized sensitivity with a specificity greater than or equal to 95%. For these threshold values, sensitivity, specificity, predictive values, and likelihood ratios were assessed. To study the impact of adding the results of the L-POCUS evaluation to several risk stratification clinical rules for pulmonary infection or sepsis (qSOFA and CRB65), AUCs were compared with or without their components with a DeLong test. For this purpose, we attributed 0, 1, or 2 points in the L-POCUS result as low, moderate, or high risk according to the predefined threshold values and assessed the AUC of the risk-stratification rules with and without adding the L-POCUS result value. Assuming a rate of death or tracheal intubation requirement of 10%, and expecting an AUC of 0.8, the number of patients required to achieve a lower limit of the 95%CI, more than 0.7, was estimated as 286. Taking into consideration that 5% of patients were not followed up or could not be evaluated, the sample size was defined as 300 patients. Missing data were not imputed. A descriptive analysis of missing data was performed and compared to the available data to assess a potential bias. All statistical analyzes were performed using STATA, version 14.2; StataCorp; College Station, TX.A total of 307 patients with suspected or confirmed COVID-19 were enrolled in this study. Among them, 2 were subsequently excluded and 9 could not be followed up (2.9%), leaving 296 patients for the main analyses , distribThe L-POCUS was performed by an emergency physician considered an expert, an advanced technician, and a novice in 32.2%, 44.3%, and 24.4% of cases, respectively. A CT-scan was performed in 170 patients (55.4%).The results of the L-POCUS are outlined in +) 1.49[95%CI: 1.4–1.6]; Negative likelihood ratio (LR-) 0; Positive predictive value (PPV) 3.9%[95%CI: 3.7–4.3]; Negative predictive value (NPV) 100%). 184 patients (62.4%) had intermediate-risk (score 1 to 15) and, among them, 4 (2.17%[95%CI: 0.8–5.5]) had an unfavorable outcome . Finally, 17 patients (5.7%) had a high-risk (score≥16) and, among them, 4(23.5%) had an unfavorable outcome at Day 14 . The proportion of patients requiring oxygen therapy was 11.6% (11/95), 33.2% (61/184) and 76.5% (13/17), in the low-risk, intermediate-risk and high-risk subgroup population, respectively.The highest L-POCUS score with a sensitivity (Se) of at least 95% was 0 point and the lowest value with a specificity (Sp) of at least 95% was 16 points. Using these cutoffs, 95 patients (32.1%) had a low-risk (score = 0) and none of them had an unfavorable outcome at Day 14 , 58 (24.2%) had a positive SARS-CoV-2 RT-PCR, and among them, 37.9% (22/58) needed oxygen therapy. At Day 14, 4 patients with confirmed COVID-19 were dead (6.9%). In this population, the AUC of L-POCUS was 0.97[95%CI: 0.92–1.00] , Panel BIn our prospective POCUSCO study of non-severe patients with confirmed or suspected COVID-19, L-POCUS had good results in predicting death or the need for invasive ventilation within the 14 days following ED admission and it appears to be a promising tool for risk stratification. However, because of a lower-than-expected rate of patients with an unfavorable outcome, the 95%CI of our estimates are wide, with an upper value of the AUC not achieving the predefined threshold qualifying clinical relevance with a good level of evidence.Based on its performance in diagnosing pneumonia and ARDS, L-POCUS ought to be a useful diagnostic and risk stratification tool in the initial assessment of suspected COVID-19 patients , 21. It et al. showed the good prognostic performances of L-POCUS to risk-stratify confirmed COVID-19 patients ) . The besTo our knowledge, POCUSCO is the largest multicentric, prospective study evaluating L-POCUS to risk-stratify COVID-19 patients. The most important limitation of this study is the low rate of the primary endpoint. On the basis of the first cohorts of COVID-19 inpatients, we anticipated a rate of mortality or invasive ventilation requirement of 10% . It was 2. Yet, obesity has been identified as a condition associated with a higher risk of worsening. Moreover, only a quarter of participating patients had a positive SARS-CoV-2 RT-PCR. The other patients may have had a minor form of COVID-19, or another less severe disease. Finally, in the absence of a derivation model, it is not methodologically justified to assess the calibration of L-POCUS Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Major commentsWhy did you choose the follow-up at 14 days and not until hospital discharge ?How did you classify the emergency physician as an expert, an advanced technician or a novice? Can you provide more details about the training?The population include mainly outpatient. Patients were not critical with only 27.7% patients who required oxygen at admission and only 2.7% met the primary outcome (death or invasive intubation). This is the main limitation of this study.It would be interesting to explore the performance of L-POCUS only for the restricted population of COVID19 that required oxygen at admission (moderate or severe patients).Minor commentsPage 6 line 89: What is the meaning of LUS (The lung ultrasound score ?).Since the aim of the study is to assess L-POCUS for COVID19 patients, could you provide the proportion of the three following features: the number (%) of i) positive SARS-CoV-2 RT-PCR, ii) typical CT-scan lesions, iii) highly-suspected COVID-19 based on the in-charge physician judgement?Reviewer #2: The authors aimed to determine whether lung POCUS is a reliable tool for identifying COVID-19 patients at risk for worsening. To answer this question, they assessed the performance of a score based on B-line intensity . I guess that the authors refers to the LUS score. I think it is necessary to name it in the manuscript.The manuscript is well written. The question is adequately introduced and the statistical analyses used seem appropriate. The outcome is robust.Nevertheless, I have two main concerns. First of all, the delay for the realization of the ultrasound (within the first 48 hours) seems to me to bring confusion. Patient with COVID-19 can evolve unfavorably during the first 48 hours following admission to the ED. This aspect restricts the range of use of this tool, especially in ED. Could the authors specify the average time between ED admission and the completion of POCUS?Second, using this score, the authors were able to identify patients who did not die or require mechanical ventilation within 14 days after ED admission. I have some concerns about its relevance in ED. Isn't 14 days far too long to be included in a ED-discharge decision strategy? Do the authors know the proportion of patients that required oxygen administration for each risk class (between ED admission and 14 days)? Could the authors discuss this point ?However, despite these remarks, I think the paper will meet the criteria for publication.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Dr. Richard Chocron, MD, PhDReviewer #1: Reviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  29 Jan 2023Dear Academic Editor, We thank you for your attention to our study entitled “Point-of-care ultrasonography for risk stratification of non-critical suspected COVID-19 patients on admission (POCUSCO): a prospective binational study” and for giving us the opportunity to improve our manuscript. Please find enclosed a point-to-point answer to the very useful comments from the Reviewers.We think that the Reviewers suggestions and the associated revisions have substantially improved the quality of the manuscript. We hope that the editorial staff and Reviewers will find this revised version suitable for publication in the “PLOS One”.Yours sincerely,On behalf of all the authors, Dr. François Morin and Prof. Pierre-Marie Roy  Response to reviewers’ letterPlease find below our point-by-point responses to all issues raised by the reviewers and the changes we made in our manuscript. Reviewer #1:Major commentsWhy did you choose the follow-up at 14 days and not until hospital discharge ?Response to reviewers: When we designed this protocol, the scientific literature, derived from patient data infected during the first COVID-19 wave, was unclear, especially which COVID-19 patients required hospitalization. However, as only non-severe patients were included, it was foreseeable that a significant proportion of the included patients could be initially managed at home or with a very short hospitalization. Nevertheless, as observed in our trial, some of them could worsen during the following days; indeed, severe pneumonia usually occurs 7 to 14 days after the first COVID-19 symptoms. Performing a systematic follow-up at Day 14 was more constraining but allowed a rigorous and similar evaluation of our main judgement criterion for all included patients. We could also have proposed an earlier follow-up at 3 days or 7 days, because what we wanted to identify by ultrasound was a subgroup of patients with a low-risk of worsening, who could be safely managed at home. We believe that a 14-day endpoint was a good trade-off. Furthermore, the same endpoint was used in some therapeutic trials .How did you classify the emergency physician as an expert, an advanced technician, or a novice? Can you provide more details about the training?Response to reviewer: We thanks the reviewer for this question, which refers to a point that was unclear in the initial version of the manuscript. We used the definition proposed by Po-Yang Tsou et al . They defined advanced ultrasonographers as clinicians with more than 7 days of training in LUS and novice sonographers as physicians with no prior experiences in ultrasound or minimal training (≤7 days) in LUS. The cutoff of 7 days of training was made by consensus among the authors based on training duration specified in the included studies. However, among the “advanced” ultrasonographers, we distinguished a group of practitioners as experts because they had a university degree in advanced lung ultrasound skills enabling them to do teaching, research, and development about ultrasound. The following precision has been added in “materials and methods” section of the manuscript : “According to Po-Yang Tsou et al., novice sonographers were defined as physicians with no prior experiences in ultrasound and no or minimal training (≤ 7 days) in lung ultrasound; advanced ultrasonographers were defined as clinicians with more than 7 days of training in LUS, and expert ultrasonographers were defined as clinicians with a university degree in advanced lung ultrasound skills enabling them to do teaching, research and development about ultrasound”. The population include mainly outpatient. Patients were not critical with only 27.7% patients who required oxygen at admission and only 2.7% met the primary outcome (death or invasive intubation). This is the main limitation of this study. It would be interesting to explore the performance of L-POCUS only for the restricted population of COVID19 that required oxygen at admission (moderate or severe patients).Response to reviewer: We thank the reviewer for this suggestion. The AUC of L-POCUS in this subgroup of patients was 0.97 [95%CI: 0.85–1.00].We added this result in the “Patients with Positive SARS-CoV-2 RT-PCR” section, as follows: “Among 240 tested patients (78.2%), 58 (24.2%) had a positive SARS-CoV-2 RT-PCR, and among them, 37.9% (22/58) needed oxygen therapy. At Day 14, 4 patients with confirmed COVID-19 were dead (6.9%). In this population, the AUC of L-POCUS was 0.97[95%CI: 0.92–1.00] . The AUC was similar in the subgroup of patients requiring oxygen therapy: 0.97[95%CI: 0.852–1.00].”Minor commentsPage 6 line 89: What is the meaning of LUS (The lung ultrasound score?).Response to reviewer: We thank the reviewer for his/her vigilance and have replaced LUS by “L-POCUS” in the whole manuscript.Since the aim of the study is to assess L-POCUS for COVID19 patients, could you provide the proportion of the three following features: the number (%) of i) positive SARS-CoV-2 RT-PCR, ii) typical CT-scan lesions, iii) highly-suspected COVID-19 based on the in-charge physician judgement?Response to reviewer : As proposed by the reviewer, the proportion of the three categories of included patients has been clarified in the manuscript, with the sentence: “Among them, 2 were subsequently excluded and 9 could not be followed up (2.9%), leaving 296 patients for the main analyses , distributed as follows: 8.2% (24/296) with positive SARS-CoV-2 RT-PCR at admission, 5.7% (17/296) with typical CT-scan lesions and 86.1% (255/296) with highly clinically suspected COVID-19 based on the in-charge physician judgement” Reviewer #2: The authors aimed to determine whether lung POCUS is a reliable tool for identifying COVID-19 patients at risk for worsening. To answer this question, they assessed the performance of a score based on B-line intensity . I guess that the authors refers to the LUS score. I think it is necessary to name it in the manuscript.Response to reviewer: We agree with this comment and have added this precision in the manuscript: “Four ultrasound aeration patterns were defined and scored 0 to 3, allowing calculation of the L-POCUS score, theoretically ranging from 0 to 36 (named Lung Ultrasound Score (LUS) by Zhao et al.) .”The manuscript is well written. The question is adequately introduced, and the statistical analyses used seem appropriate. The outcome is robust.Nevertheless, I have two main concerns. First of all, the delay for the realization of the ultrasound (within the first 48 hours) seems to me to bring confusion. Patient with COVID-19 can evolve unfavorably during the first 48 hours following admission to the ED. This aspect restricts the range of use of this tool, especially in ED. Could the authors specify the average time between ED admission and the completion of POCUS?Response to reviewer: We agree with this comment. The entire team, on behalf of all the inclusion centers, gives you the assurance that the vast majority of L-POCUS were carried out at the emergency department admission. Unfortunately, we are not able to provide the proportion of L-POCUS performed at ED admission or later within the 48h. Indeed, we did not record the time of the ultrasonography. However, in the centers participating to the study, few medical wards dedicated to care of COVID-19 patients were equipped with an ultrasonography device. Therefore, it is unlikely that an important proportion of patients included in the study had their ultrasonography performed more than 24h after admission to the ED. This point has been added in the limitations section of the manuscript. Second, using this score, the authors were able to identify patients who did not die or require mechanical ventilation within 14 days after ED admission. I have some concerns about its relevance in ED. Isn't 14 days far too long to be included in a ED-discharge decision strategy? Do the authors know the proportion of patients that required oxygen administration for each risk class (between ED admission and 14 days)? Could the authors discuss this point ?Response to reviewer: We agree with this comment about the relevance of this tool in the context of a discharge strategy. However, when the protocol was drafted (February 2020), scientific evidence suggested that the onset time of COVID-19-related ARDS was 8–12 days, which was inconsistent with ARDS Berlin criteria, which defined a 1-week onset limit . It therefore seemed relevant to choose Day 14 because this time allowed us to know the evolution of all patients, including those who would consult within the first hours of onset of their symptoms and who would have a late adverse course. Patients usually consult in the emergency department a few days after the onset of symptoms (median delay between symptom onset and ED admission of 8 days in our study). In these patients, an unfavorable evolution in the hours or first days after admission is therefore likely and screening by pleuropulmonary ultrasound to assess this short-term risk is therefore quite interesting, especially if their RT-status COVID PCR is known (AUC 0.97[95%CI: 0.92–1.00]).Do the authors know the proportion of patients that required oxygen administration for each risk class (between ED admission and 14 days)? Could the authors discuss this point ?Response to reviewer: We thank the reviewer for this suggestion and we mentioned the proportion of patients requiring oxygen therapy for each risk class as follow: “ The proportion of patients requiring oxygen therapy was 11.6% (11/95), 33.2% (61/184) and 76.5% (13/17), in the low-risk, intermediate-risk and high-risk subgroup population, respectively.” A discussion of this point has been added in the discussion section: “The rate of patients requiring oxygen therapy, at any time of their management, was proportional to the L-POCUS risk category. In comparison with low-risk category (11%), the rate of oxygen therapy was 3-fold higher in intermediate-risk and 7-fold higher in high-risk L-POCUS category. This result strongly suggest that L-POCUS signs correspond to lung lesions.” In addition to this, as mentioned in the response to the first reviewer, the analyze of the performance of L-POCUS in the restricted population of positive COVID-19 patients that required oxygen at admission has been added: “Among 240 tested patients (78.2%), 58 (24.2%) had a positive SARS-CoV-2 RT-PCR, and among them, 37.9% (22/58) need oxygen therapy. At Day 14, 4 patients with confirmed COVID-19 were dead (6.9%). In this population, the AUC of L-POCUS was 0.97[95%CI: 0.92–1.00] . The AUC was similar in the subgroup of patients requiring oxygen therapy: 0.97[95%CI: 0.852–1.00].”AttachmentResponse to reviewers_POCUSCO_PLOSOne_16012023.docxSubmitted filename: Click here for additional data file. 10 Apr 2023Point-of-care ultrasonography for risk stratification of non-critical suspected COVID-19 patients on admission (POCUSCO): a prospective binational studyPONE-D-22-28983R1Dear Dr. Morin,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: The authors have responded to my suggestions and comments in a satisfactory manner. I have no further comments to make.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Prunus cerasus (sour cherry) extract (PCE) on atherosclerosis-associated cardiac dysfunction, described by the impairment of the NO-PKG (nitric oxide–protein kinase G) pathway and the antioxidant capacity. Initially, a rabbit model of atherosclerotic cardiovascular disease was established by administering a cholesterol-rich diet, enabling the examination of the impact of 9 g/kg PCE on the pre-existing compromised cardiovascular condition. After that, the animals were divided into four groups for 12 weeks: the (1) untreated control group; (2) PCE-administered healthy rabbits; (3) hypercholesterolemic (HC) group kept on an atherogenic diet; and (4) PCE-treated HC group. Dyslipidemia, impaired endothelial function, and signs of diastolic dysfunction were evident in hypercholesterolemic rabbits, accompanied by a reduced cardiac expression of eNOS , PKG, and SERCA2a . Subsequent PCE treatment improved the lipid profile and the cardiac function. Additionally, PCE administration was associated with elevated myocardial levels of eNOS, PKG, and SERCA2a, while no significant changes in the vascular status were observed. Western blot analysis further revealed hypercholesterolemia-induced increase and PCE-associated reduction in heme oxygenase-1 expression. The observed effects of anthocyanins indicate their potential as a valuable addition to the treatment regimen for atherosclerosis-associated cardiac dysfunction.This study evaluates the potential therapeutic effects of anthocyanin-rich  Despite the improving the treatment of cardiovascular diseases, the overall incidence of heart failure (HF) is still increasing ,2,3. Cor2+) homeostasis with the help of the sarcoplasmic reticulum Ca2+-APTase (SERCA2a), as well as regulating the giant elastic protein called titin [Patients with diastolic dysfunction (DD) suffer from impaired left ventricle (LV) relaxation and filling during the cardiac cycle. These abnormalities are characterized by enhanced ventricular stiffness due to alterations in both the extracellular matrix (ECM) and the cardiomyocytes . Evidenced titin .Metabolic comorbidities such as diet-induced hyperlipidemia and obesity are important contributors to atherosclerotic cardiovascular diseases, in which oxidative stress and inflammatory processes cause endothelial dysfunction observed also in the coronary microvasculature and affect NO bioavailability . Thus, dIn recent years, anthocyanins, a subgroup of flavonoids, have garnered increasing interest in the medicinal field due to their antioxidant and anti-inflammatory properties related to their chemical structure . The maiThe scientific literature suggests that low-dose-cholesterol-fed rabbits are valuable models of dyslipidemia, atherosclerosis, and cardiac dysfunction. These experimental animals are very susceptible to an atherogenic diet—at least in part—as this type of diet can upregulate cholesteryl ester transfer protein (CETP) expression ,28. AddiThe authors of this publication consider it important to note that in the animal model used, the disease under investigation had already been developed; thus, we were interested in therapeutic rather than preventive options. Anthocyanins are often used for preventing and delaying the onset of a disease; however, we examined them as treatment options focusing on managing and alleviating the underlying causes of the existing compromised cardiovascular condition.Prunus cerasus L.) extract on cardiac dysfunction in a rabbit model of atherosclerotic cardiovascular disease.The primary objective of the present study is to elucidate the therapeutic effects of anthocyanin-rich sour cherry . HDL-C levels showed a similar trend (control vs HC: p = 0.0022). Results of the atherogenic index of plasma (AIP) in rabbits from the HC group were significantly higher than those calculated in the healthy control animals (p < 0.0001). In HC rabbits treated with PCE (HC + PCE), a significant improvement in the lipid profile was observed compared to the HC group. The PCE administration significantly reduced the levels of both total cholesterol and LDL-C in the HC + PCE animals, versus the HC group . Moreover, AIP values significantly decreased in the anthocyanin-treated HC rabbits, compared to the animals in the HC group (p = 0.0028). Additionally, no significant differences were detected in the serum triglyceride contents of the experimental groups. It was further noted that the CRP concentration was four-fold higher in the serum of the hypercholesterolemic rabbits compared to the healthy groups and four-fold lower in the HC + PCE animals, versus the non-treated HC group. No perceptible changes were identified in the cardiac biomarker CK-MB levels. Concerning glucose levels and liver enzymes (AST and ALT), we observed no marked changes. As a limitation of the study, the levels of NT-pro-BNP were below the detection limit in all treatment groups.Evaluated lipid parameters and cardiac biomarkers are shown in p < 0.0001), deceleration time (DecT) and isovolumetric relaxation time (IVRT) increased , and E/A ratio and e’/a’ ratio decreased  in comparison to healthy rabbits. The average E/e’ ratio observed in the HC animals was significantly higher compared to the control group (p = <0.0001), estimating an increase in the left ventricle (LV) filling pressure in the non-treated HC group. We detected a significant improvement in the echocardiographic parameters representing the diastolic function in the PCE-treated group (HC + PCE) versus the HC rabbits (p < 0.0001), the prolongation of the DecT was reduced (p < 0.0001), and Tissue Doppler Imaging (TDI) revealed a significant increase in the e’/a’ ratio (p = 0.0002) in comparison to the non-treated HC group. The PCE-treated animals exhibited a significant decrease in E/e’ ratio compared to the non-treated rabbits kept on an atherogenic diet (p < 0.0001). No significant differences were noted in the results of the Tei-index of the experimental groups (p > 0.05). Despite of the long-term hypercholesterolemia, the HC group did not exhibit any indications of deteriorated left ventricle systolic function. Heart rate (HR), ejection fraction (EF), and fractional shortening (FS) remained in the normal range in all cases. Systolic myocardial velocity (s’) and mitral annular plane systolic excursion (MAPSE) values were unaffected by both the atherogenic diet and the anthocyanin administration. The speckle-tracking echocardiographic technique showed a reduced global longitudinal strain (GLS) in the HC group in comparison to the controls, and restored GLS values were observed in the HC + PCE-treated rabbits. Last but not least, no remarkable differences were found in the left ventricle outflow tract (LVOT) parameters, including mean and maximal outflow velocities  and pressure gradients .The results of the cardiac ultrasound are summarized in  rabbits . In hypeThe contractions of the aortic rings evoked by noradrenaline (NA) were significantly lower in the HC rabbits in comparison to the controls at 1 µmol/L NA concentration. In all the groups, acetylcholine (Ach), up to 1 µmol/L concentration, elicited relaxation, while higher Ach concentrations caused contraction instead. As expected, the in vivo cholesterol treatment statistically significantly decreased the acetylcholine-evoked relaxation of aortic rings with the preservation of the acetylcholine-evoked contraction . The in vivo PCE treatment did not influence the vascular response to Ach in a statistically significant manner . Thus, rp < 0.0001) (p > 0.05). The histological sections of the HE-stained aortas are demonstrated in  0.0001) . No signp = 0.0190). Similarly, in the case of PKG and SERCA2a levels, significant differences were observed between the control and the non-treated HC groups . In HC + PCE rabbits, the expression of eNOS, PKG, and SERCA2a proteins was significantly enhanced compared to the group kept only on a cholesterol-rich diet . The left ventricle tissue expression of four decisive proteins of cardiovascular homeostasis is shown in p < 0.0001). The enhanced HO-1 expression was attenuated by PCE administration, as a significant difference was observed between the HC and HC + PCE groups (p < 0.0001). Additionally, for all proteins of interest, no significant differences were observed between the two healthy groups (control vs. C + PCE).Western blot analysis also revealed that the level of HO-1 was significantly higher in the myocardium of rabbits maintained on an atherogenic diet, compared to the expression detected in the healthy control group , fractional shortening (FS), and systolic myocardial velocity (s’) remained in the normal range. In the HC group, markedly decreased E/A and e’/a’ ratios and increased deceleration time (DecT) and isovolumetric relaxation time (IVRT) were detected, suggesting incomplete left ventricle (LV) relaxation and thus limited LV filling . MoreoveSerum analyses were conducted to evaluate parameters that may function as diagnostic indicators for estimating the severity of cardiovascular diseases. The results revealed marked dyslipidemia induced by the atherogenic diet, as the atherogenic index and the total cholesterol, LDL-C, and HDL-C levels were significantly elevated in the hypercholesterolemic group, relative to the healthy control rabbits. These outcomes are directly in consonance with previous data gathered by other authors . HoweverScientific evidence shows that dietary cholesterol induces dyslipidemia and atherosclerosis, in which oxidative stress and inflammatory processes cause endothelial dysfunction . Under aPrunus cerasus extract attenuated the atherosclerosis-associated diastolic dysfunction through the upregulation of the NO-cGMP-PKG pathway and the downstream signaling target SERCA2a in the myocardial tissue, without affecting the vascular homeostasis.As the deterioration of the NO-cGMP-PKG pathway is a main pathophysiological mechanism that contributes to diastolic impairment , the WesThe stress protein heme oxygenase-1  plays a prominent role among the defensive systems responsible for restoring the homeostasis of the cells and exerting cytoprotective activities against oxidative stress and inflammation . These fIn the present report, the mechanism behind the HO-1-downregulating effect of the PCE is not entirely clear, but it may be related to complex interactions between the anthocyanins and the cholesterol metabolism. A likely explanation for this outcome is the alleviated myocardial oxidative stress due to the significant cholesterol-lowering effect of the anthocyanins in the PCE-treated hypercholesterolemic rabbits. As HO-1 expression depends on the duration and severity of hypercholesterolemia, its level may be reduced by improving the lipid profile. Alternatively, the anthocyanins may interact with other signaling pathways in the heart that affect HO-1 expression; therefore, further investigations are needed to fully understand the possible underlying mechanisms.Prunus cerasus L.) cultivar called "Érdi bőtermő". The fruit samples were harvested from the orchard of the Research and Consulting Institute for Fruitgrowing (Újfehértó) between June and July of 2020. After the sampling, the fruits were immediately transferred to the laboratory and stored at -20 °C until use.The anthocyanin-rich extract was made from a Hungarian sour cherry  rabbits  with a body weight range of 2500–3000 g. The animals received humane care in accordance with the "Principles of Laboratory Animal Care" by EU Directive 2010/63/EU and all aspects of the experiments were approved by the University of Debrecen Committee of Animal Welfare (4/2022DEMÁB). The animals were kept in individual cages under standard conditions (a 12:12 h dark/light cycle at a temperature of 24 °C). During a 2-week-long adaptation period, the rabbits were fed normal chow and had free access to tap water.At the beginning of the study, the experimental animals were randomly divided into two groups: (1) a healthy control group which received normal rabbit chow and (2) a hypercholesterolemic (HC) group in which rabbits were kept on intermittent cycles of an “atherogenic” diet with periods of a normal diet for 32 weeks. The “atherogenic” chow contained 1% cholesterol and 1% saturated fat and was formulated by Pro Drug L.P.  ,55.Prunus cerasus extract  for 12 weeks [n = 7 in each) in the study were as follows: (1) the untreated control group received normal chow; (2) the healthy control with PCE treatment (C + PCE) received normal chow and 9 g/kg PCE dissolved in tap water; (3) the hypercholesterolemic (HC) group was kept on atherogenic chow; and (4) the PCE-treated diseased group (HC + PCE) received atherogenic chow and 9 g/kg PCE dissolved in the drinking water. The control and the HC groups were treated with vehicle. The weight of the animals was recorded once a week, and for the two groups drinking anthocyanin solution (C + PCE and HC + PCE), it was freshly prepared every day. A total of 9 g of the PCE contained 100 mg anthocyanins [After 32 weeks of creating the rabbit model of atherosclerotic cardiovascular disease, we made four subgroups from the two original ones described previously and then started the administration of the anthocyanin-rich 12 weeks ,57, everocyanins  and the Before the administration of the PCE, the assessment of the lipid profile of each rabbit was performed. At the endpoint, blood sample collection and echocardiographic measurements were carried out. The animals were sacrificed under deep anesthesia . After thoracotomy, the distal part of the thoracic aorta was excised to perform ex vivo vascular assays. The cardiac samples and the remaining part of the thoracic aorta were immediately frozen in liquid nitrogen and stored at −80 °C for Western blot analysis or in a 10% formalin solution for histology.Before and after the 12-week-long treatment, blood samples were collected from the marginal ear veins of each rabbit into BD Vacutainer SST II Advance Tubes , after 12 h fasting. The serum levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), glucose, aspartate transaminase (AST), and alanine transaminase (ALT) were determined. Specific markers like C-reactive protein (CRP), creatine kinase MB isoform (CK-MB), and N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) were measured, and the atherogenic index of plasma  was calculated as well. All parameters were recorded in the Department of Laboratory Medicine at the University of Debrecen by using automated clinical laboratory analyzers .At the endpoint, cardiac ultrasound was carried out using a Vivid E9 ultrasound machine  with a high-frequency probe (12S-D) . Rabbits2 and 5% CO2 . The Krebs solution contained (in mmol/L): NaCl: 118, KCl: 4.7, CaCl2: 2.5, NaH2PO4: 1, MgCl2: 1.2, NaHCO3: 24.9, glucose: 11.5, and ascorbic acid: 0.1, dissolved in redistilled water. The isometric contractile force of the circulatory muscle layer was measured using a transducer  connected to a WS-DA-02 workstation (MDE Research) with SPEL Advanced Isosys software version 2.9 .After sacrificing the rabbits, the distal part of the thoracic aorta was isolated, and 2 mm wide rings were cut off . The rings were mounted horizontally at a 10 mN resting tension using a wire instrument  in a 4-chamber isolated organ bath system with 10 mL vertical chambers . The chambers were filled with Krebs solution oxygenated with 95% O50) was determined for each aortic ring. After a 60 min wash-out, the EC50 of noradrenaline was administered to each ring and, after the stabilization of the contractile force, an acetylcholine (Ach) E/c curve was constructed (from 1 nmol/L to 1 mmol/L).After a 60 min incubation period, a noradrenaline (NA) concentration–response (E/c) curve (from 1 nmol/L to 10 µmol/L) was generated, and the half-maximal effective noradrenaline concentration .The responses of aortic rings obtained from the same animal were averaged. The effect of noradrenaline was defined as the maximal phasic (quick) increase in the vascular tension beyond the resting level (adjusted previously to 10 mN). In turn, the effect of acetylcholine was defined as the percentage decrease in the vascular tonic (stabilized) tension produced by the ECThe Western blot technique was used to identify proteins from the left ventricle (LV). Firstly, for protein isolation, 300 mg of deep-frozen LV tissue samples were homogenized in 800 µl buffer solution ), using a disperser . Then, the homogenates were centrifuged at 2000 rpm for 10 min at 4 °C. The supernatant contained the cytosolic and mitochondrial fractions. All samples were incubated on ice for 1 h in a solution containing 0.1% Triton-X-100 . After a second centrifugation at 14000 rpm for 20 min at 4 °C, the resulting supernatants were used as the cytosolic–mitochondrial extracts. From 10 μL of the samples, the total protein concentration was measured with the help of a spectrophotometer , using the BCA assay . Next, 50 μL of the residual volume was diluted with Laemmli buffer , and finally, the remaining samples were stored at −80 °C.® blot scanner  were used. The analysis of the scanned Western blots was carried out using Image Studio Digits ver. 5.2. software . In all cases, the normalization to the background and the standardization to a housekeeping protein (GAPDH) were conducted. Finally, data analysis was performed on three independent experiments.Samples mixed with Laemmli buffer were separated via SDS–polyacrylamide gel electrophoresis (SDS-PAGE)  in a running buffer, and then proteins were transferred onto a nitrocellulose membrane  in a transfer buffer at 25 V for 90 min. After 1 h of blocking with 5% BSA solution , blots were incubated overnight at 4 °C with primary antibodies: anti-glyceraldehyde-3-phosphate-dehydrogenase ; anti-endothelial nitric oxide synthase ; anti-protein kinase G ; anti-sarco/endoplasmic reticulum calcium ATPase 2a ; and anti-heme oxygenase-1 . The following morning, the membranes were washed with TBS-T for 3 × 10 min before being incubated with horseradish–peroxidase (HRP)-conjugated secondary (anti-mouse or anti-rabbit) antibodies. To identify and detect protein bands, enhanced chemiluminescent substrate  and LiCor C-DigitTo visualize tissue structure, aortic samples were fixed in 10% neutral buffered formalin (pH = 7.4) for 24 h. The following day, tissue samples were washed in water for 1 h and then stored in 70% ethanol until further steps. After dehydration and clearing with xylene, from the final paraffin-embedded block 5 μm thick slices were sectioned, deparaffined, and stained with hematoxylin–eosin (HE). For image acquisition, a Nikon Eclipse 80i microscope  was used. From the cross-sectioned aortic samples the thickness of the intima and the media was measured using the length-measuring function of the Nikon NIS-Elements BR (Ver5.41.00) software , followed by the calculation of the average intima/media ratios. p) were less than 0.05 (p < 0.05). All data are expressed as means ± standard error of the means (SEM).Data analysis was performed using GraphPad Prism software for Windows, version 8.00 . The D’Agostino–Pearson test was used first to determine the Gaussian distribution. According to the result of the normality test, further statistical analysis was carried out using either one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis or  the Kruskal–Wallis test followed by Dunn’s post-test. Results were considered to be significantly different when the probability values (Prunus cerasus extract (PCE) attenuated the already existing cardiac diastolic dysfunction induced by an atherogenic diet without influencing the vascular status. This experiment also adds to the growing body of research showing that HO-1 overexpression may act as a double-edged sword in the maintenance of cell homeostasis. PCE administration restored the diastolic dysfunction observed in the untreated hypercholesterolemic animals and induced the upregulation of the cardioprotective eNOS, PKG, and SERCA2a proteins. Taking into consideration the positive results of the PCE treatment, incorporating dietary anthocyanin supplementation could offer a promising therapeutic approach in the treatment of cardiac dysfunction linked to atherosclerosis.The primary outcome of the recent study is that the upregulation of the cardiac NO-PKG pathway along with SERCA2a induction as a result of a 12-week-long treatment with


MicroRNA-155 (miR-155) is a multifunctional miRNA whose expression is known to be involved in a range of physiological and pathological processes. Its association with several oral diseases has been established. However, the specific role of miR-155 in orthodontic tooth movement remains unclear. In this study, we investigated the impact of miR-155 on osteoclast differentiation and orthodontic tooth movement models, aiming to explore the underlying mechanisms.In this experiment, we utilized various agents including miR-155 mimic, miR-155 inhibitor, as well as non-specific sequences (NC mimic & NC inhibitor) to treat murine BMMNCs. Subsequently, osteoclast induction (OC) was carried out to examine the changes in the differentiation ability of monocytes under different conditions. To assess these changes, we employed RT-PCR, Western blotting, and TRAP staining techniques. For the orthodontic tooth movement model in mice, the subjects were divided into two groups: the NaCl group  and the miR-155 inhibitor group (injected with AntagomiR-155). We observed the impact of orthodontic tooth movement using stereoscopic microscopy, micro-CT, and HE staining. Furthermore, we performed RT-PCR and Western blotting analyses on the tissues surrounding the moving teeth. Additionally, we employed TargetScan to predict potential target genes of miR-155.During osteoclast induction of BMMNCs, the expression of miR-155 exhibited an inverse correlation with osteoclast-related markers. Overexpression of miR-155 led to a decrease in osteoclast-related indexes, whereas underexpression of miR-155 increased those indexes. In the mouse orthodontic tooth movement model, the rate of tooth movement was enhanced following injection of the miR-155 inhibitor, leading to heightened osteoclast activity. TargetScan analysis identified SOCS1 as a target gene of miR-155.Our results suggest that miR-155 functions as an inhibitor of osteoclast differentiation, and it appears to regulate osteoclasts during orthodontic tooth movement. The regulatory mechanism of miR-155 in this process involves the targeting of SOCS1.The online version contains supplementary material available at 10.1186/s12903-023-03443-8. Orthodontic tooth movement occurs as a result of mechanical force exerted on the teeth, leading to remodeling of the periodontal membrane and alveolar bone. When the appropriate orthodontic force is applied, the periodontal membrane transmits this force to the alveolar bone. This transmission of force causes bone resorption on the side experiencing pressure. It is widely recognized that this step is considered the rate-limiting step in orthodontic tooth movement . EnhanceMicroRNAs (miRNAs) are a group of non-coding small RNA molecules, typically composed of 20–22 nucleotides. They play a crucial role in post-transcriptional gene regulation by targeting specific genes. MiRNAs bind to the 3’-untranslated region (3’-UTR) of target genes, resulting in reduced gene expression at the post-transcriptional level. This binding can lead to mRNA degradation or transcriptional inhibition, thereby regulating post-transcriptional gene expression . MicroRNMicroRNA-155 (miR-155) is a multifunctional miRNA known to be involved in a wide range of physiological and pathological processes. Its expression changes have been associated with the regulation of hematopoietic lineage differentiation, immune response, inflammation, tumor development, viral infections, cardiovascular diseases, Down syndrome, and more , 14. CurCurrently, some researchers have demonstrated the inhibitory effect of miR-155 on the regulation of osteoclast differentiation . However6 cells per well, using α-MEM medium supplemented with 10% FBS and 30ng/ml macrophage colony-stimulating factor (M-CSF) . After 2 days of culture at 37 °C and 5% CO2, the BMMNCs appeared as small and round cells under the microscope. Osteoclastogenesis was initiated by adding receptor activator of nuclear factor-κB ligand (RANKL) at a concentration of 100ng/ml  to the medium.Bone marrow-derived monocytes (BMMNCs) were obtained from 6-week-old male C57BL/6 mice and cultured. The experiments were conducted in accordance with the ARRIVE  guidelines and the approved institutional guidelines for animal research set by the Animal Care and Use Committee of the Beijing Stomatological Hospital, Capital Medical University, Beijing, China (KQYY-202008-005). Mice were euthanized by inhalation of an overdose of isoflurane, and their bodies were immersed in 75% alcohol for 10 min. The femur and tibia from the lower limbs were isolated and washed with Phosphate Buffered Saline (PBS) containing Penicillin-Streptomycin solution (PS). The marrow was flushed out with medium until the marrow cavity turned white. The collected suspension was centrifuged at 1100 rpm for 6 min, and the supernatant was discarded. The cells were resuspended in cell suspension by adding 20% FBS α-MEM medium. The cell suspension was then incubated at 37 °C and 5% CO2 for 24 h. After incubation, the supernatant was aspirated from the culture dish. The cells were seeded in 6-well plates at a density of 1–2 × 10All miR-155 transfection reagents used in this experiment were purchased from RiboBio  Co., Ltd. A volume of 5 µl of 20µM miR-155 mimic and NC mimic was diluted in 120 µl of 1X riboFECT™ CP Reagent Buffer. For miR-155 inhibitor and NC inhibitor, a volume of 10 µl of 20µM stock solutions was gently mixed and diluted. Subsequently, a volume of 12 µl of riboFECT™ CP Reagent was added to the mixture, gently blown and mixed, and incubated at room temperature for 0–15 min. The resulting mixture was then added to 6-well plates containing PS-free medium to initiate cell transfection. The final concentration of the miRNA mimic was 50nM, and the final concentration of the miRNA inhibitor was 100nM. After 48 h, the total RNA of the cells was extracted. Some cells were switched back to 10% FBS α-MEM medium, and osteoclast-inducing factors  were added for 3 or 5 days, during which the medium was changed once.Twenty-four 6-week-old C57BL/6 male mice, weighing approximately 20 g, were obtained from SPF (Beijing) Biotechnology Co., Ltd. All experiments were conducted in accordance with animal ethics guidelines. The mice were randomly divided into four groups: TM 5d + NaCl (5 days of orthodontics tooth movement with NaCl injection), TM 12d + NaCl (12 days of orthodontics tooth movement with NaCl injection), TM 5d + AntagomiR-155 (5 days of orthodontics tooth movement with AntagomiR-155 injection), and TM 12d + AntagomiR-155 (12 days of orthodontics tooth movement with AntagomiR-155 injection) (n = 6/group). Three days before and on the day of orthodontic treatment, submucosal injections of 50 µl normal saline or 10nmol AntagomiR-155 dissolved in 50 µl saline were administered at four sites around the right maxillary first molar: buccal mesial-distal and palatal mesial-distal sites. During the second administration, the force was applied to the right maxillary first molar using a spring fixed to the upper incisor, which served as the anchorage teeth to mesially move the right maxillary first molar. At the end of the force application, the maxilla or the gingiva surrounding the mobile teeth was harvested from the animal model for subsequent analysis.After the animals were euthanized, the maxillary specimens were collected and fixed in a 4% paraformaldehyde solution. The tooth movement distance of the first molar in each specimen was observed using stereoscopic microscopy. The Image J software was utilized to measure the shortest distance between the first molar and the second molar under the microscope. Three measurements of the same sample, performed by the same researcher, were averaged.Micro-CT scanning was conducted on the tissue samples from various groups at the Medical Imaging Research and Testing Laboratory of Capital Medical University. Data Viewer, CT Vox, and CT An software were employed to measure the tooth movement distance. Specifically, the shortest distance between the crowns of the first and second molars in the sagittal direction was measured. Three measurements of the same sample, performed by the same researcher, were averaged.The animal tissues were decalcified using 10% EDTA in PBS (pH 8.0) at 4℃. After decalcification, the samples were dehydrated and embedded in paraffin. Sections of 5 μm thickness were obtained and stained with hematoxylin and eosin. The alveolar bone was examined using a light microscope .TRAP staining was performed on tissue sections from different groups in vivo and on osteoclasts in vitro after 5 days of induction. The staining procedure followed the standard protocol of the TRAP staining kit . Osteoclasts were identified as TRAP-positive cells with three or more nuclei, displaying a purplish-red color under the microscope. Following staining, five random fields were selected from each group for observation and counting using a light microscope . Statistical analysis was conducted to analyze the results.Three days after osteoclast induction of BMMNCs, total RNA was extracted from the cells using Trizol reagent . The gingival tissue from the animal model was also isolated using Trizol reagent after sufficient grinding. Reverse transcription was carried out following the manufacturer’s protocol . RT-PCR reactions were performed using the SYBR Green PCR kit . The primer sequences for osteoclast-related markers are listed in Table Cells and gingival tissues were lysed using radioimmunoprecipitation (RIPA) lysis buffer and protease inhibitors  to extract proteins. The protein extraction protocol has been previously described . PrimaryFor IHC analysis, paraffin-embedded sections were successively deparaffinized in xylene, dehydrated in ethanol, and incubated in citrate buffer  for 10 min at 90 °C for antigen repair. Then, the sections were detected for antigens using a Cell and Tissue staining kit . Sections were incubated at 4 °C with anti-SOCS1  staining overnight. Images were obtained using a fluorescence microscope system .Statistical analysis was performed using SPSS 22.0 software. Student’s t-test was used to determine statistical significance. A p-value less than 0.05 was considered to indicate a significant difference.To investigate the role of miR-155 in the osteoclast differentiation of monocytes, BMMNCs from mice were treated with M-CSF (30ng/ml) and RANKL (100ng/ml) to induce osteoclast formation. The expression of osteoclast-related markers, including Cathepsin K (CTSK), TRAP, and Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), was examined by RT-PCR. Additionally, the expression of miR-155 was also evaluated. The results showed that compared with BMMNCs without induction, the expression of osteoclast-related markers  was significantly increased, while the expression of miR-155 was significantly decreased after 3 days of osteoclast induction. These findings indicate an inverse relationship between miR-155 expression and osteoclast-related indicators  Fig. A, D-E. AWe collected gingival tissue surrounding the right upper first molar from different groups of animal models and performed RT-PCR to analyze gene expression. On the fifth day of force application, the AntagomiR-155 group (TM 5d + AntagomiR-155) exhibited increased expression of miR-155 compared to the orthodontic group (TM 5d), along with elevated expression of osteoclast-related markers CTSK and TRAP , confirming the presence of target binding sites in the 3’ UTR region of SOCS1 Fig. . MeanwhiConversely, a miR-155 inhibitor was employed to downregulate miR-155 expression, and the transfection efficiency was verified after 48 h. The findings revealed a substantial decrease in miR-155 expression in the miR-155 inhibitor group, accompanied by an opposite trend in SOCS1 expression, showing a significant increase Fig. C. After In addition, the expression of SOCS1 in gingival tissue from the animal model was explored. The results demonstrated a statistically significant difference observed on the 12th day, the expression of SOCS1 was significantly increased in the AntagomiR-155 group Fig. E. ImmunoOverall, these findings provide evidence that miR-155 directly targets SOCS1 to modulate osteoclast differentiation both in vitro and in vivo. The expression pattern of SOCS1 is inversely regulated by miR-155, consistent with the expression of other osteoclast-related markers.MiRNAs play a crucial role as post-transcriptional regulators in maintaining bone homeostasis. In our study, we initially observed an inverse relationship between miR-155 expression and osteoclast-related markers during osteoclast differentiation, suggesting that inhibition of miR-155 may enhance osteoclast activity. This study is the first to directly manipulate miR-155 expression in BMMNCs using both overexpression and low expression reagents, allowing for positive and negative verification and obtaining relatively stable in vitro experimental results.To further investigate the impact of miR-155 on osteoclast differentiation, we designed a mouse orthodontic tooth movement model. Osteoclast activation is a critical step in orthodontic tooth movement, and therefore, we examined the effect of miR-155 on this process. Following the injection of the miR-155 inhibitor, we observed an accelerated tooth movement rate, providing further confirmation of our hypothesis that miR-155 acts as an inhibitor of osteoclasts.The average duration of orthodontic treatment in clinical practice is approximately 20 months. However, as the treatment time extends, both doctors and patients may encounter increased challenges and risks. These include problems such as root resorption, tooth demineralization, and even dental caries . TherefoPrevious studies have indicated that miRNAs possess several favorable characteristics, including their small molecular weight, charge, good water solubility, and rapid degradation and renal metabolism within the body . Based oIn this study, we utilized 6-week-old C57BL/6 male mice as the experimental subjects to establish an orthodontic tooth movement model and investigate the role of miR-155 in vivo. Local injection of AntagomiR-155 at the recommended concentration, as stated in the product manual, was found to accelerate orthodontic tooth movement, potentially through the promotion of local osteoclast activity. MiR-155 may be implicated in the inhibition of osteoclast differentiation. Nonetheless, it is important to acknowledge the limitations of this study. For instance, the duration of the in vivo experiment was relatively short, and the investigation did not extend to the assessment of alveolar bone remodeling and tooth stability following tooth movement. Jiang et al. demonstrated a decrease in miR-155 expression in the gingival crevicular fluid of orthodontic patients as the degree of root resorption increased . HoweverRegarding the investigation of miR-155 target genes, we initially identified a target binding site between the 3’UTR region of SOCS1 and miR-155 using TargetScan software. Subsequently, we performed RT-PCR and western blot to examine the changes in SOCS1 expression upon transfection of cells with miR-155 overexpression or low expression, and we used the Immunohistochemistry to observe the changes of SOCS1 around the orthodontic teeth after injection of miR-155 inhibitor. The results revealed that the expression of SOCS1 exhibited an opposite trend to that of miR-155, consistent with the expression pattern of osteoclast-related markers, and the immunohistochemistry result in this study verified the spatial expression of SOSC1 in the tissues during the tooth movement. Suppressor of cytokine signaling 1 (SOCS1) serves as a critical negative regulator for various inflammatory cytokines. It functions as the most potent member of the SOCS family, exerting negative regulation on the JAK/STAT pathway, and plays a crucial role in suppressing the secretion of proinflammatory cytokines such as interferon-γ (IFN-𝛾) . The JAKIn conclusion, this study has revealed the inhibitory effect of miR-155 on osteoclast differentiation, with enhanced differentiation observed when miR-155 was suppressed. It has been demonstrated that miR-155 directly targets the 3’UTR region of SOCS1, leading to the inhibition of SOCS1 expression and subsequent regulation of osteoclast differentiation. These findings suggest that miR-155 and SOCS1 could serve as potential targets for controlling osteoclast-mediated orthodontic tooth movement.Below is the link to the electronic supplementary material.Supplementary Material 1